The ability of nucleic acids to bind their complementary sequences is the basis of assays for the detection of specific nucleic acid sequences. An available nucleic acid, the probe, can be modified to facilitate it's detection or it's separation from other nucleic acids. Various kinds of labels have been used. Radioactive or fluorescent labels can be introduced enzymatically or chemically. Enzymes can be chemically coupled to nucleic acid to generate the probe. Particular nucleic acid sequences can be coupled to a probe to facilitate it's detection by methods based on that sequence—e.g. polymerase reaction, ligase chain reaction, Q-beta replicase amplification, etc.
Generally, the target of such assays is a particular nucleic acid that represents a small fraction of the total sample. Consequently assay sensitivity is an important consideration and the various labels and methods have been designed with a view to attaining high sensitivity.
Conceptually in a simple hybridization assay two strands of complementary nucleic acid come together to form a double stranded structure. The probe strand contains a label and the target strand is detected by means of the labeled strand to which it becomes attached by hybridization.
The assay sensitivity depends on the amount of label captured in the hybridization complex. The label may be something that can be detected directly—e.g. a flourescent moiety or a colored substance (chromogen). The label may be something that can be detected indirectly. This would include a radioactive isotope, or particular nucleic acid template or sequence which functions as a ligand or as a target for subsequent hybridization to a different labeled nucleic acid. The label attached to the nucleic acid can be a chemical moiety to which something else binds which can itself be detected directly or indirectly. For example if the probe is modified by attachment of an antigen then specific antibody molecules can bind the antigen. Such antibodies may be detected directly if appropriately labeled e.g. with fluorescent moieties or indirectly after reaction with a second appropriately labeled antiserum. Either the first or second antiserum may be labeled with an enzyme e.g. alkaline phosphatase or peroxidase, the catalytic activity of which further enhances assay sensitivity. In addition to antigens, other moieties that bind to proteins may be used to label the probe. Biotin labeled probes can be detected after reaction with appropriately labeled avidin or streptavidin. Proteins that bind specific nucleic acid sequences can be used either alone or appropriately labeled or as part of fusion proteins with a partner which is itself detectable either directly or indirectly. Nucleic acids can be labeled by enzymatically or chemically attaching fluorescent or colored molecules which can be directly detected. Particles, enzymes and polymeric substances or crystals with appropriate surface modifications can be used as direct or indirect labels for nucleic acids.
Both chemical and enzymatic methods are routinely used to place a label in the probe sequence. Short oligo-nucleotides are often labeled at the 5′ end by using T4 polynucleotide kinase to introduce a radioactive phosphate. The 3′ end is often labeled with T4 RNA ligase or with terminal transferase. Chemical modifications permit a great variety of labels to be introduced internally or at the ends of synthetic or naturally occurring nucleic acids. Large DNA molecules can be labeled internally by first reacting with bisulfite to generate amino residues to which NHS esters of labels (e.g. fluorescent dyes) can be readily coupled. A common way to introduce labels makes use of the nick translation activity of E. coli Pol I holoenzyme. The Klenow fragment of Pol I primed by short random oligo-nucleotides ( e.g. hexamers or nanomers) provides a convenient and robust labeling method.
Whether the ultimate signal detection is direct or indirect, the amount of signal is dependent on the amount of label attached per unit mass of the probe molecules (for radioactive probes this is referred to as specific activity of the probe). There are practical limitations to how much signal-generating moiety can be incorporated into probe molecules without inhibiting assay performance. Labels that result in chemically modified nucleic acid, whether incorporated chemically or enzymatically, typically interfere with hybridization characteristics of a probe if more than a few percent of the nucleotide residues are modified. Presumably bulky side groups destabilize duplexes. A high concentration of label moieties can change the physical and chemical properties of the molecule, increasing susceptibility to cleavage as occurs with radioactive probes or increasing stickiness for example as occurs with fluorescent labeled probes. This can result in poor hybridization or in high non-specific binding both of which limit assay sensitivity.
This invention is a method for modifying the DNA sequence prior to or during labeling so as to promote network formation during hybridization with the result that each target sequence becomes attached to many labeled probe molecules. The assay sensitivity is thereby increased. This can be applied to many kinds of hybridization assay. Particular applications include hybridization to nucleic acid arrays, in situ hybridization, dot blots, Southern blots, Northern blots, sandwich hybridization assays etc.